Significant amounts of peroxidatively-active substances, such as blood or hemoglobin, are not normally present in bodily fluids. Such substances, particularly hemoglobin and its derivatives, often indicate abnormal bleeding which is symptomatic of various conditions, even cancer. To provide for early diagnosis, bodily fluids are often assayed to determine the presence, absence, or concentration of peroxidatively-active substances in bodily fluids.
In a diagnostic assay, a peroxidatively-active substance, like hemoglobin and its derivatives, causes a reaction between a hydroperoxide and an oxidizable spectrophotometric substrate or dye. The hydroperoxide releases oxygen which oxidizes the spectrophotometric substrate to yield a detectable change, e.g., a color change indicating the presence of the peroxidatively-active substance. In a quantitative assay, the intensity of that change may be used to determine the concentration of the peroxidatively-active substance in the bodily fluid test sample. Typical bodily fluids assayed include, for example, cerebral fluid, feces, gastrointestinal fluids, ocular fluid, saliva, serum, spinal fluid, vomit, or urine.
The presence of hemoglobin or blood in bodily fluids is generally termed "occult blood." When present, occult blood is not always apparent from visual examination. Occult blood may result from hemorrhages, tumors, ulcers, inflammations, disease or other injury of a particular organ or organs. Detecting occult blood permits diagnosis of a hemorrhage, other injury, or disease in, for example, the stomach, intestines, or urinary tract. The presence of blood in urine, for example, can indicate kidney or urinary tract damage. Normally, urine does not contain detectable amounts of blood.
Occult blood can appear in a bodily fluid as intact red blood cells or as free hemoglobin. In some situations, hemolysis of the red blood cells occurs after the cells have entered a bodily fluid, e.g., urine. Bodily fluid samples containing red blood cells also contain some hemolyzed occult blood.
Hemoglobinuria is the presence of free hemoglobin in the urine without red blood cells. In general, free hemoglobin from plasma is excreted by the kidney into the urine. Hematuria, in contrast to hemoglobinuria, is the presence of intact red blood cells in urine. Hematuria indicates a specific defect in the microscopic functional unit (the nephron) of the kidney. Hematuria may also indicate bleeding in the kidney, the ureter, the bladder or the urethra. Presently, differentiating between blood as cells versus free hemoglobin is generally considered to be of little diagnostic significance.
In urine, free hemoglobin may indicate that blood cells have ruptured due to traumatic passage through the kidney and urinary tract to the bladder. Urinary hemoglobin may also indicate that blood cells have been exposed to and hemolyzed by dilute urine in the bladder. Urinary hemoglobin, therefore, may indicate renal disorders, infectious diseases, neoplasms, or traumas to the urinary tract. More specifically, urinary hemoglobin can indicate a transfusion reaction, hemolytic anemia, or paroxysmal hemoglobinuria, or result from poisonings or severe burns. A positive urinary hemoglobin assay, without the presence of red cells, can indicate myoglobinuria as a result of traumatic muscle injury. Assays for urinary hemoglobin provide a vehicle for early diagnosis of such conditions.
Another peroxidatively-active substance, myoglobin, the red respiratory pigment of muscle tissue, is also not normally found in bodily fluids. Myoglobin is similar to hemoglobin in its composition and chemical reactions. Muscular injury can cause myoglobin to be discharged from muscle cells, to circulate in the plasma, and then be excreted in the urine. In addition, certain genetic muscle disorders can cause loss of myoglobin, which subsequently appears in the urine. Myoglobin may also be found in the urine after a cardiac infarct. The presence of myoglobin in urine is known as myoglobinuria. Assays capable of detecting myoglobin in bodily fluids, such as urine, can therefore provide significant diagnostic benefit.
As discussed, the presence of a peroxidatively-active substance in urine suggesting hematuria, hemoglobinuria, or myoglobinuria depends upon the nature and/or severity of the specific disorder, disease, or injury. Assays may also be used to test bodily fluids for other peroxidatively-active substances, such as leukocytes and bacteria. Overall, detecting a peroxidatively-active substance in a bodily fluid assists the early diagnosis of many disorders, diseases, injuries, and infections.
Traditionally, dry phase reagent strips, or dipsticks, have been used as assays for peroxidatively-active substances, as well as other analytes, in bodily fluids. A typical dry phase test strip is commercially available from Miles, Inc. under the trademark HEMASTIX.RTM..
A reagent strip generally contains one or more test pads, having a porous carrier matrix, such as a paper matrix, impregnated with a buffered mixture of an organic hydroperoxide and an indicator dye, affixed to a plastic strip or handle. The reagent strip is dipped into a bodily fluid sample to test for the presence of analytes such as hemoglobin, or another peroxidatively-active substance. Exemplary dry phase reagent strips are described in U.S. Pat. Nos. 3,986,833; 3,917,452; 3,975,161; 4,587,220; 4,755,472; 5,318,894; and 5,362,633.
By changing color, a test pad indicates the presence of an analyte, such as hemoglobin or myoglobin, in the test sample. The color change intensity may be proportional to the concentration of the peroxidatively-active substance in the sample. Comparing the resulting test pad color to a standardized chart, an analyst can subjectively determine, the amount of an analyte, such as a peroxidatively-active substance, present in the sample.
Dry phase reagent strips, however, suffer from a number of disadvantages. Assays using reagent strips are quite labor intensive. As noted above, reagent strips require an analyst to conduct the test and match the color change with a chart and subjectively determine the quantity of an analyte in a test sample. Not only is this time consuming, but more importantly, it introduces the possibility of human error. In addition, a reagent strip often requires the results be read within a certain amount of time after testing. This creates an additional time pressure on an analyst and increases the possibility for human error and misdiagnosis. Finally, having multiple test pads on a single reagent strip exposes the possibility of cross-contamination of reagents during manufacture or testing. This possibility may compromise the reagent strip's diagnostic integrity.
A need exists, therefore, for an accurate assay of bodily fluids to test for peroxidatively-active substances. The assay should be capable of qualitative and/or quantitative analysis. The assay should be useable in clinical settings such as a clinical laboratory and in non-clinical settings such as a doctor's office or even at home. The assay should provide clear, reproducible detection and measurement of the peroxidatively-active substances in the bodily fluid permitting more accurate diagnosis and medical treatment. Use of the assay should be time efficient and have a low likelihood of error.